If a neutrally charged particle, such as a biological cell, is placed in a uniform electric field, such as provided by a pair of same-size planar electrodes, the biological cell does not move toward either one electrode or the other because the attractive forces from both electrodes are the same.
On the other hand, if a neutrally charged biological cell is placed in a non-uniform electric field, such as provided by two electrodes which are both not planar, as shown in PRIOR ART FIG. 1, the biological cell forms a dipole, is attracted to one electrode with greater attractive force than the other, and moves towards the electrode having the greater attractive force.
Such a use of a non-uniform electric field is used in dielectrophoresis, and the concept of using dielectrophoresis to align living cells, followed by a fusion/electroporation pulse, to fuse cells has been in the literature since early 1970's. This process is used to produce hybrids of two different cell types for therapeutic purposes, for hybridoma production for producing monoclonal antibodies, for nuclear fusion, and for producing other hybrid cells.
Dielectrophoresis is the process of applying an electrical force on neutrally charged particles such as living cells. The electrical force causes adjacent living cells to be compressed against one another, as shown in FIG. 5. The force from dielectrophoresis (dielectrophoretic force) results from applying a non-uniform electric field, produced by an electrode pair to which a voltage is applied. The non-uniform electric field separates charges (ions) inside the cells forming a dipole. After the dipole has been formed, the non-uniform electric field then moves the cells towards the highest or lowest electric field intensity. This movement is dependent on the relative conductivities and permittivities of the medium and the biological cells or particles. The living cells are also aligned in the non-uniform electric field, as shown in PRIOR ART FIG. 2.
The dielectrophoretic force is a function of the electric field squared, so electric field polarity is not important. The dielectrophoretic force is also a function of the relative conductivities and permittivities of the medium and the particles or cells. The conductivities and permittivities are a function of the frequency of the applied electric field. Typically, an AC voltage wave, such as a sine wave, is applied across electrodes to produce an alternating electric field. The sine wave voltage, frequency, and duration are optimized for specific cell types.
After the AC wave is applied to align and compress the cells, one or more fusion/electroporation pulses are applied to permeabilize adjacent cell membranes (form pathways between adjacent cell membranes) and to cause cell membranes from both adjacent cells to fuse or commingle. These pathways permit the contents of the cells to mix forming a hybrid fused cell.
Permeabilization is conventionally done in electric fields having uniform electric field intensity so that all cells in the electric field are permeabilized in a uniform manner. The uniform electric field is achieved by using parallel flat plate electrodes.
On the other hand, it is known that permeabilization of all cells in an electric field that has non-uniform electric field intensity would result in the cells being permeabilized in a non-uniform manner. Such non-uniformity in permeabilization is undesirable. Fewer pathways form in the cell membranes resulting in fewer cell fusions.
Following the fusion pulses, another AC field can be applied to hold the cells together while the fused cells stabilize (mature). In some cases, the AC voltage has been linearly increased or decreased to prevent damage to the cells due to a sudden application of a field.
The published PCT International Application No. WO 03/020915 A2 describes AC waveforms that can be applied at a low level to align the cells without creating large forces producing turbulence. After the cells are aligned, the waveform then applied provides a large force which compresses the cells creating a large mutual surface area between the cells just before the permeabilization electric field pulse is applied.
Examples of cell fusion applications include hybridoma production and nuclear transfer. A recent application for electrofusion is to produce therapeutic hybrids for cancer immunotherapy. These hybrids are produced from cancer tumor cells and immune system dendritic cells in an ex vivo process. Each treatment requires a large number of viable hybrids, which results in a new requirement for high efficiency in the hybrid production process. Commercial and clinical uses of these techniques are now important requiring large numbers of hybrid products to be produced in a single batch.
There are a number of techniques (electrical, mechanical, and chemical) available to perform cell fusion. This invention relates to electrical means. The current electric art uses a voltage waveform generator connected to an electrode device or chamber. With respect to known electrical, mechanical, and chemical techniques, the following U.S. patents are of particular interest and are incorporated herein by reference:
U.S. Pat. No. 4,326,934 Apr. 27, 1982 Pohl
U.S. Pat. No. 4,441,972 Apr. 10, 1982 Pohl
U.S. Pat. No. 4,578,168 Mar. 25, 1986 Hofmann
U.S. Pat. No. 4,695,547 Sep. 22, 1987 Hillard
U.S. Pat. No. 4,699,881 Oct. 13, 1987 Matschke et al
U.S. Pat. No. 4,764,473 Aug. 16, 1988 Matschke et al
U.S. Pat. No. 4,784,954 Nov. 15, 1988 Zimmermann
U.S. Pat. No. 4,804,450 Feb. 14, 1989 Mochizuki
U.S. Pat. No. 5,007,995 Apr. 16, 1991 Takahashi
U.S. Pat. No. 5,304,486 Apr. 19, 1994 Chang
From the above, it is known to use electrodes or chambers that produce non-uniform electric fields. One such example is two coaxial electrodes forming a chamber. The coaxial chamber was described in detail by Pohl in a book published in 1978. The coaxial chamber was discussed in relation to theoretical dielectrophoresis considerations.
Nevertheless, there has been no description of how to effectively set the dimensions of the coaxial chamber for any particular application. Cell fusion using electrical means requires a non-uniform electric field to align and compress the cells and a uniform electric field to permeabilize the cells. To provide the highest possible efficiency in producing the fused hybrid cells, as required in commercial and clinical applications, the geometric dimensions of the chamber must be carefully selected.
Initially in any cell fusion process one must bring the cells into alignment and contact. In any case, sufficient force must be applied to each cell to overcome the negative surface charge. As stated above, merely applying a uniform electric field will not move a cell because the net charge of the cell is zero. Thus, from the definition of electric field, there is no force applied, because the charge equals zero:Force=(Electric Field)*(Charge)
However, a non-uniform field induces the positive ions inside each cell to move to one side and the negative ions to move to the opposite side producing a dipole, as shown in PRIOR ART FIG. 1. Once the dipole is induced, because of the presence of a non-uniform electric field, a net force is exerted on the cell because the intensity of the field is greater on one side than the other. The movement of cells in one direction causes the cells to align. Since the cells are now dipoles, the negative side of one cell will attract the positive side of another cell overcoming the negative surface charge, as shown in PRIOR ART FIG. 2. The non-uniform electric field is produced by the electrode device or chamber. The non-uniformity is a function of the electrode configuration, examples of which are shown in PRIOR ART FIGS. 1 and 2.
Generally, the cell types to be fused are placed in a low conductive medium (for example 100 microsemens/cm) to minimize ohmic heating that may harm the cells and that causes turbulence in the medium, thus reducing the number of fused hybrids. In this respect, it would be desirable for biological cells being subjected to cell fusion to be treated so as to reduce heating during cell alignment and cell membrane contact.
The waveform generator has multiple functions. The first function is to produce the AC voltage waveform that is converted into an AC field by the electrode pair or chamber. This AC field brings the cells into alignment/contact. The second function is to compress the cells by briefly increasing the amplitude of the AC waveform. The third function is to produce a pulse voltage that produces an electric field that electroporates the membranes of the cells in close contact, fusing the cells. The fourth function is to apply a low amplitude AC voltage to hold the cells in alignment until the fusion products become viable or stable (mature).
One of the factors for successful fusion is the membrane contact between the adjacent cells. The closer this contact before the fusion pulse is applied, the higher the efficiency of fusion. In U. Zimmermann, et al., “Electric Field-Induced Cell-to-Cell Fusion”, J. Membrane Biol. 67, 165-182 (1982), Zimmermann points out that increasing the AC wave electric field strength just before the fusion pulse may be the optimum approach. Clearly, it would be desirable for biological cells that are to undergo cell fusion to be pretreated with pre-fusion non-linear electric field waveforms to produce sufficient force to bring about increased cell membrane contact and then to immediately apply a uniform electric field pulse(s) that permeabilizes the cell membranes in contact, thereby leading to cell fusion.
It would be very desirable to have a chamber that will produce a large number of fused products by applying a large force (proportional to a non-uniform electric field) on the adjacent cells to compress the cells to create a larger surface area between them and then to immediately apply a uniform electric field from one electrode to the next that will permeabilize the largest number of cell membranes in contact.
It is also desirable to have a chamber of sufficient volume to produce a large number of hybrid products.
In view of the above, it would also be desirable to produce a chamber with sufficient uniform and non-uniform electric fields to provide the largest number of fused hybrid cells.
Thus, while the foregoing body of prior art indicates it to be well known to use coaxial chambers, the prior art described above does not teach or suggest a method to determine how to select the chamber geometry which has the following combination of desirable features: (1) provides sufficient force (non-uniform field intensity) to compress the cells providing a large membrane contact area without excessive heating; (2) provides sufficient uniform field intensity to permeabilize the cells; and (3) produces a large number of hybrid products. The foregoing desired characteristics are provided by the unique coaxial cell fusion chamber of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.
Additional U.S. patents and published U.S. patent applications that are of interest include:
U.S. Pat. No. 4,561,961 Dec. 31, 1985 Hofmann
U.S. Pat. No. 5,001,056 Mar. 19, 1991 Snyder et al
U.S. Pat. No. 5,589,047 Dec. 31, 1996 Coster et al
U.S. Pat. No. 5,650,305 Jul. 22, 1997 Hui et al
US2003/0082163, May 1, 2003 Shu
Additional literature references include:    1. R. Bischoff, et al., “Human Hybridoma Cells Produced by Electro-Fusion”, Fed. Eur. Biochem. Soc. Lett. 147, 64-68 (1982).    2. L. Changben, et al., “Use of Human Erythrocyte Ghosts for Transfer of 125.sub.I-BSA and 125.sub.I-DNA into Animal Cells from Cell Fusion”, Scientia Sinica (Series B) 25, 680-865 (1982).    3. C. S. Chen, et al., “Biological Dielectrophoresis: The Behavior of Lone Cells in a Non-uniform Electric Field”, Ann. N.Y. Acad. Sci. 238, 176-185 (1974).    4. Coster, H. G. L. and Zimmermann, U. “Direct Demonstration of Dielectric Breakdown in the Membranes of Valonia utricularis.” Zeitschrift fur Naturforschung. 30 c, 77-79.1975.    5. Coster, H. G. L. and Zimmermann, U. “Dielectric Breakdown in the Membranes of Valonia utricularis: the role of energy dissipation”. Biochimica et Biophysica Acta. 382, 410-418,1975.    6. Coster, H. G. L. and Zimmermann, U. “The mechanisms of Electrical Breakdown in the Membranes of Valonia utricularis.” Journal of Membrane Biology. 22, 73-90,1975.    7. K. Kaler, et al., “Dynamic Dielectrophoretic Levitation of Living Individual Cells”, J. Biol. Phys. 8, 18-31 (1980).    8. A. R. Murch, et al., “Direct Evidence that Inflammatory Multi-Nucleate Giant Cells Form by Fusion”, Pathol. Soc. Gr. Brit. Ire. 137, 177-180 (1982).    9. Neumann, Bet al. “Cell Fusion Induced by High Electrical Impulses Applied to Dictyostelium”, Naturwissenschaften 67, 414, 1980    10. Petrucci, General Chemistry: Principles and Modern Applications, 4th ed., p. 621, 1985 (no month).    11. Zimmermann et al., Electric Field-Induced Cell-to-Cell Fusion, The Journal of Membrane Biology, vol. 67, pp. 165-182 (1982) [no month].    12. Pohl, H. “Dielectrophoresis”, Cambridge University Press, 1978.    13. H. A. Pohl, “Biophysical Aspects of Dielectrophoresis”, J. Biol. Phys. 1, 1-16 (1973).    14. H. A. Pohl, et al., “Continuous Dielectrophoretic Separation of Cell Mixtures”, Cell Biophys. 1, 15-28 (1979).    15. H. A. Pohl, et al., “Dielectrophoretic Force”, J. Biol. Phys. 6, 133 (1978).    16. H. A. Pohl, et al., “The Continuous Positive and Negative Dielectrophoresis of Microorganisms”, J. Bio. Phys. 9, 67-86 (1981).    17. Sale, J. H. and Hamilton, W. A. “Effects of High Electric Fields on Micro-Organisms”, Biochimica et Biophysica Acta. 163, 37-43, 1968.    18. Sepersu, E. H., Kinosita, K. and Tsong, T. Y. “Reversible and Irreversible Modification of Erythrocyte Membrane Permeability by Electric Fields” Biochimica et Biophysica Acta. 812, 779-785, 1985.    19. J. Vienken, et al., “Electric Field-Induced Fusion: Electro-Hydraulic Procedure for Production of Heterokaryon Cells in High Yield”, Fed. Eur. Biomed. Soc. Lett. 137, 11-13 (1982).    20. H. Weber, et al., “Enhancement of Yeast Protoplast Fusion by Electric Field Effects”, A Preprint for Proceedings of the Fifth International Symposium on Yeasts,London, Ontario, Canada, Jul. 80.    21. Zimmermann, U. “Electrical Field Mediated Fusion and Related Electrical Phenomena”, Biochimica et Biophysica Acta. 694, 227-277. 1982.    22. Zimmermann, U. et al “Fusion of Avena Sativa Mesophyll Proptoplasts by Electrical Breakdown”, Biochimica et Biophysica Acta. 641, 160-165, 1981. 1982.    23. U. Zimmermann, et al., “Electric Field-Induced Release of Chloroplasts from Plant Protoplasts”, Naturwissen 69, 451 (1982).    24. U. Zimmermann, et al., “Electric Field-Mediated Cell Fusion”, J. Biol. Phys. 10, 43-50 (1982).    25. U. Zimmermann, “Cells with Manipulated Functions: New Perspectives for Cell Biology, Medicine, and Technology”, Angew. Chem. Int. Ed. Engl. 20, 325-344 (1981).    26. Electromechanics of Particles, Thomas B. Jones, 1995, Cambridge University Press.    27. Electroporation and Electrofusion in Cell Biology, Eberhard Neumann, Arthur E. Sowers, and Carol A. Jordon, Plenum Press, New York 1989.
As explained below with respect to the subject invention, prior art ratios r1/r2 and gaps of known prior art chambers are outside the respective ranges of the subject invention. Such prior art are as follows:                1. Dielectricophoresis of cell size liposomes, 13 December 1993. r1/r2=0.25, gap=0.75 mm.        2. Hofmann U.S. Pat. No. 4,578,168, Mar. 25, 1986, r1/r2=0.139, gap=0.155 mm.        3. Hillard U.S. Pat. No. 4,695,547, Sep. 22, 1987, r1/r=0.162, gap=13 mm        4. Matschke U.S. Pat. No. 4,699,881, Oct. 13, 1987, r1/r2=0.98, gap=0.4 mm.        5. Zimmerman U.S. Pat. No. 4,764,473, Aug. 16, 1988, no dimensions.        6. Mochizuki, U.S. Pat. No. 4,804,450, Feb. 14, 1989, r1/r2=0.962, gap=2 mm.        7. Takahashi, U.S. Pat. No. 5,007,995, Apr. 16, 1991, r1/r2=0.263, gap=2.8 mm        8. Chang, U.S. Pat. No. 5,304,486, Apr. 19, 1994, r1/r2 not given, gap=0.5 to 2.0 mm        9. Shu, US2003/0082163, May 1, 2003, r1/r2 not given, gap 2 to 5 mm.        